DIAZOTIZED [I125] DIIODOSULFANILIC ACID AS A LABEL FOR CELL-SURFACE MEMBRANES - STUDIES ON ERYTHROCYTES

1. 1. Diazotized 2,6-diiodosulfanilic acid (DDISA) appears to have properties suitable to serve as an artificial, non-penetrating label of cell surface membranes. Therefore, the conditions for selective labeling of cell surface membranes as compared to intracellular proteins as well as a method for its chemical determination were explored in the present study. 2. 2. DDISA reacts with α-naphthol at neutral pH to produce a compound (1-hydroxy-4-(2,6-diiodo-4-sulfo-1-phenylazo-(naphthylene)), DSPN) with a characteristic spectrum in the visible range (Amax 430 nm). The absorbance of the reaction product, DSPN, is linearly proportional to the concentration of DDISA and can be used as a method for the colorimetric determination of DDISA. Reaction of DDISA with a molar excess of α-naphthol was also used as a method for inactivating unreacted DDISA to terminate labeling prior to cell fractionation. 3. 3. [125I]DDISA reacts avidly with a variety of basic, neutral and acidic proteins as well as with cell membranes to form an acid-stable covalent azo linkage. 4. 4. Effectiveness of labeling of the surface membrane of intact erythrocytes after incubation with [125I]DDISA was assessed by the ratio of 125I incorporated into membrane proteins compared to intracellular proteins. When intact erythrocytes were exposed to [125I]DDISA, the optimal labeling of membranes occurred at 37°C after 20 min of incubation time and at a concentration of 10-4 M [125I]DDISA in the incubation media. Under these conditions the ratio. © 1979.